The discovery of regulatory small RNAs is among the most significant biomedical breakthroughs in recent history. To date, three major classes of small RNAs have been identified, namely the microRNA (miRNA), the small-interfering RNA (sRNA), and the piwi-interacting RNA (piRNA). These classes differ in their biogenesis, their length, and their tissue distribution (Ghildiyal, M., and Zamore, P. D. (2009). Nat Rev Genet 10, 94-108; Kim, V. N. et al (2009). Nature reviews 10, 126-139). miRNAs are ˜22 nucleotides (nt) in length and are generated from hairpin-shaped primary transcripts by two sequential processing steps mediated by a nuclear (Drosha) and a cytoplasmic (Dicer) RNAse III endonuclease. miRNAs are loaded in RISC (RNA-induced silencing complex), which main component is Argonaute 2 (Ago2), to mediate degradation and/or translation block of specific target messenger RNAs (mRNAs) via Watson-Crick base pairing partial sequence complementarity (Fabian, M. R., et al. (2010). Annual review of biochemistry 79, 351-379). miRNAs are ubiquitinously expressed and control a wide range of cellular activities, including development, immune function, and cell death (Fabian, M. R., et al. (2010). Annual review of biochemistry 79, 351-379). siRNAs derive from double stranded-RNAs that are processed by Dicer into 19 nt small RNAs. siRNAs are also incorporated in RISC to post-transcriptionally silence specific target mRNAs in stem-cells (Ghildiyal, M., and Zamore, P. D. (2009). Nat Rev Genet 10, 94-108). piRNAs derive from single stranded-RNAs that are processed by a poorly understood mechanism into 24-30 nt small RNAs. piRNAs mediate both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ cells (Ghildiyal, M., and Zamore, P. D. (2009). Nat Rev Genet 10, 94-108). Importantly, recent analyses of data coming from next generation sequencing technologies, also known as deep or high-throughput sequencing, from cell cultures and tissues have revealed the existence of other classes of small RNAs, including those from sno-RNAs and tRNAs or from Alu repeats. Overall, these findings suggest that cells may generate a wide range of regulatory small RNAs with a broad variety of processing mechanisms and functions.
The inventors have explored whether atherogenic stimuli regulate the expression of novel regulatory small RNAs, which in turn may modulate apoptosis and inflammation in macrophages. Atherosclerosis is caused by an artery wall thickening, also known as lesion, as a result of the accumulation of lipids, cells, and extracellular matrix in the area between the endothelium and the underlying smooth muscle cells (Moore, K. J., and Tabas, I. (2011). Cell 145, 341-355). A key initiating step of atherosclerosis is the subendothelial accumulation of ApoB-LPs (apolipoprotein B-containing lipoproteins) composed of cholesteryl fatty acyl esters and triglycerides, which are transported in the blood as LDL (Low-density lipoprotein) (Moore, K. J., and Tabas, I. (2011). Cell 145, 341-355). Activation of endothelial cells by ApoB-LPs leads to the recruitment of macrophages, which play a major role all along atherosclerosis from the early phases of lesion formation to the advanced ones. Indeed, in early phases, macrophages engulf lipoproteins and lipids, and become the so-called foam cells, which secrete inflammatory cytokines and undergo apoptosis. Rapid efferocytic clearance of the apoptotic foam cells leads to suppression of the inflammatory response ultimately retarding lesion progression. However, in advanced lesions, macrophage apoptosis is not properly coupled with phagocytic clearance leading to the necrotic plaque formation. The build-up of necrotic debris ultimately promotes inflammation, plaque disruption, and eventually thrombosis. Both macrophage death rate and efficiency of apoptotic cell clearance are processes that control lesion progression (Moore, K. J., and Tabas, I. (2011). Cell 145, 341-355). Induction of macrophage apoptosis in atherosclerotic lesions involves the chronic and cumulative effect of several features including oxidant stress, cytokines, oxidized LDL (oxLDL), activation of Fas death pathway, saturated fatty acids, and endoplasmic reticulum stressors (Moore, K. J., and Tabas, I. (2011). Cell 145, 341-355). Therefore, understanding the molecular mechanisms underlying apoptosis in macrophages upon atheroma-relevant stimuli is a key goal in deciphering lesion progression.
The inventors have shown that, in macrophages stimulated with atherogenic lipids, RNYs (also called “Y RNAs”) are processed into small RNAs which are about 24-34 nucleotide (nt) long and are referred to as small-RNYs (s-RNYs). These s-RNYs cause the degradation of a subset of mRNAs crucial for atherogenesis and ultimately modulate apoptosis and inflammatory response in lipid-laden macrophages.
RNYs are ˜110 nt long cytoplasmic, non-coding RNAs that are characterized by extensive base-pairing of the 5′ and 3′ regions and by the association with the proteins Ro60 and La/SSB to form the Ro ribonucleoprotein complex. In macrophages incubated with atherogenic stimuli, such as palmitic acid (PA), generation of s-RNYs requires Ago2 and the single-stranded RNA binding protein hnRNP A1 which is the most abundant core proteins of the ribonucleoprotein complex. hnRNP A1 shuttles to the cytoplasm to directly bind RNYs and modulate the positioning/recruitment of Ago2 which in turn participates into the catalysis and maturation of RNYs into s-RNYs.
s-RNYs regulate pro-inflammatory and pro-apoptotic pathways in lipid-laden macrophages by controlling the expression levels of a subset of transcripts which are critical in regulating the pathogenesis of atherosclerosis, such as Fos, KLF2, and Nr4a1. Importantly, s-RNYs are responsible, at least in part, for the activation of p38 and NF-κB signaling pathways in PA-treated macrophages, ultimately promoting cell death and inflammatory response.
In accordance the above finding that s-RNYs are the main small RNAs regulating immune response in the pathogenesis of atherosclerosis, a significant increase of s-RNY expression was demonstrated in mouse models for atherosclerosis and in the serum of a cohort of patients with stable coronary artery disease (CAD). Furthermore, sRNY expression levels in CAD patients were positively correlated with pro-atherogenic lipids and inflammatory condition, while negative association was found with atheroprotective HDL.
Altogether, the inventor's results indicate that s-RNY expression can be used as a diagnostic marker and a therapeutic target for atherosclerosis.